Characterization of resistance genes and replicon typing in Carbapenem-resistant Klebsiella pneumoniae strains

Objective In our study, K. pneumoniae strains (non-susceptible to carbapenem) (n = 60) were obtained from various clinical samples from Rize State Hospital between 2015 and 2017 and it is aimed to identify antibiotic resistance genes and replicon typing. Methods Antibiotic susceptibility tests of the strains were performed with Kirby-Bauer disk diffusion test and the Vitek-2 automated system (BioMerieux, France). Antibiotic resistance genes and replicon typing was characterized by PCR method. Results It was determined that K. pneumaniae isolates were mostly isolated from the samples of the intensive care unit. All of the K. pneumoniae strains examined in this study were found to be ampicillin/sulbactam and ertapenem resistant but colistin susceptible. Amoxacillin/clavulonic acid resistance was detected at 98.14% of strains. The blaOXA-48 gene was mostly detected in isolates. The most common type of plasmid was I1 and 3 different plasmid types were found in five different strains together. Conclusion This study also shows that the distribution of NDM-1 and OXA-48 carbapenemases has increased since the first co-display in Türkiye and that IncHI1 is the first record in our country. This study provides an overview of the major plasmid families occurring in multiple antibiotic-resistant strains of K. pneumoniae. To our knowledge, this study represents the first report of IncHI1 record in Türkiye.


Background
The increase in antimicrobial resistance is a concern for human and animal health all over the world.Globally, 700.000 people die each year from infections caused by antimicrobial resistance [1,2].Klebsiella pneumoniae is a gram-negative opportunistic pathogen that causes healthcare-associated infections in hospitalized and immunocompromised individuals [1].In neonates, it is one of the leading causes of sepsis in patients with hematological malignancies [3,4].It may colonize the gastrointestinal tract, skin, nose and throat of healthy individuals [5].It has recently acquired additional genetic features, the number of severe infections due to K. pneumoniae increased and treatment efficacy decreased due to the emergence of hypervirulent (HV) or antibiotic-resistant K. pneumoniae strains [6].K. pneumoniae has a high resistance to many antibiotic groups, such as beta-lactam antibiotics, fluoroquinolones, and aminoglycosides [7].Antimicrobial resistance is commonly associated with the spread of infectious plasmids and the acquisition of resistance genes that normally occur by horizontal gene transfer and can also carry virulence factors [8].The increase in the clinical use of carbapenems has led to the emergence of carbapenem-resistant K. pneumoniae worldwide in recent years.Carbapenem-resistant K. pneumoniae is mainly associated with metallo-β-lactamases, such as K. pneumoniae carbapenemase (KPC), oxacillinase-48 (OXA-48), and New Delhi metallo-β-lactamase (NDM), imipenemase (IMP), and Verona integron-encoded metallo-β-lactamase (VIM).Although the prevalence of these plasmid-mediated carbapenemases in the world varies geographically, it has been reported many times that OXA-48 producers are the source of nosocomial outbreaks since their first detection in Türkiye [9].
The emergence of carbapenem-resistant K. pneumoniae strains with increased virulence has made it difficult to distinguish between these strains and hypervirulent strains.Therefore, the increasing association between carbapenem resistance and hypervirulence in K. pneumoniae is alarming [10].Horizontal gene transfer via plasmids plays an important role in increasing bacterial antibiotic resistance (AMR).The identification of plasmid properties and their relationship to different bacterial hosts provides information about the contribution of plasmids to AMR delivery.Molecular identification of these plasmid and progeny genotypes provides an understanding of the difference between the expression of AMR genes by plasmids and the expression of these genes by bacteria [11].Plasmid typing can provide insight into the epidemiology of resistance plasmids [12].In particular, PCR-based replicon typing (PBRT) is frequently used for typing plasmids [11].In Enterobacterales, including K. pneumoniae, there are 28 plasmid types that can be distinguished by PBRT [13].
Our study aimed to identify non-carbapenem-susceptible K. pneumoniae strains sent from various clinics in Rize State Hospital (RSH) in 2015-2017, which were found to have developed resistance in recent years and to determine their antibiotic resistance profiles and responsible genes for resistance.Plasmid typing was performed by examining replicon types.Since RSH functions not only as a local but also as a regional hospital.The antibiotic resistance profile of K. pneumoniae in various clinical samples, the detection of the resistance genes, and the examination of the spread of these genes have potential effects on infection control, public health, and rational antibiotic use.

Obtaining and identifying isolates
Sixty K. pneumoniae strains isolated from patients treated at Rize State Hospital between 2015 and 2017 were included in the study.Samples were stocked at -20 °C and − 80 °C to contain 20% glycerol.Identification of strains were identified and verified by conventional methods and the Vitek-2 automated (BioMerieux, France).

Antimicrobial susceptibility test
Antibiotic susceptibility tests were determined by Kirby-Bauer disk diffusion test and the Vitek-2 automated system (BioMerieux, France).This information was taken retrospectively from the hospital information management system.For antibiotic susceptibility testing: amikacin, amoxicillin-clavulanic acid, ampicillin/sulbactam, cefepime, cefotaxime, ciprofloxacin, colistin, ertapenem, imipenem, meropenem, piperacillin/tazobactam, tigecycline, gentamicin discs were studied at 18-24 °C for 18 h of plaques.Inhibition zone diameters around the antibiotic discs were measured following the incubation of antibiotics, and the results were evaluated according to the Clinical and Laboratory Standards Institute (CLSI) criteria [3].

Stocking of K. pneumoniae isolates and total DNA isolation
K. pneumoniae isolates were grown in 4 mL of antibioticfree Luria-Bertani (LB) medium and grown in a shaking incubator at 37 °C overnight.By taking 800 µL of bacterial suspensions, 20% glycerol stocks were prepared.1000 µL of the remaining bacterial suspensions were centrifuged at 14,000 rpm for 5 min.The pellets were dissolved in 1000 µL of distilled water and boiled for 10 min.After centrifugation at 14,000 rpm for 10 min, 500 µL of the supernatant was used as DNA source [4].

Screening of antibiotic resistance genes in K. pneumoniae strains by PCR
Antibiotic resistance genes (bla TEM , bla SHV , bla CTX-M1 , bla CTX-M2 , bla GES , bla VEB , bla PER , bla KPC , bla IMP , bla VIM , bla NDM , bla OXA-51 , bla OXA-58 , bla OXA-23 , bla OXA-40 , and bla OXA-48 ) of K. pneumoniae strains isolated from hospital infections were characterized by PCR method.Primers in Table 1 were used to search for genes responsible for antibiotic resistance by PCR. 5 µL of genomic DNA, 1 µL of each primer, 5 µL of reaction buffer, 3 µL of 25 mM MgCl 2 , 2.5 µL of 4 mM dNTP and 0.2 µL of Taq Polymerase were added to a reaction mixture and the final volume was made up to 50 µL with distilled water.PCR results will be analyzed on 1% agarose containing 0.5 mg/L ethidium bromide and then visualized under UV light.As the PCR conditions, the conditions specified in the relevant references in Table 1 were used.Amplicons of the size specified in the reference studies were accepted as positive.

Determination of replicon types
Plasmid analysis in K. pneumoniae strains was investigated by PCR method.Specific primers for HI1, HI2, I1, X, L/M, N, FIA, FIB, W, Y, P, FIC, A/C, T, FIIs, FrepB, and K/B replicons by Carattoli [7].Replicon typing was performed with (Table 2).Primers, target DNA sequence and PCR fragment size to be obtained are as in Table 2. PCR; 2.5 µL of 10X Buffer, 1.5 µL of 25 mM MgCl 2 , 1.25 µL of 4 mM dNTPs, 1 µL of each primers (10 pmol), 0.2 µL of 5 U/µL Taq DNA pol was prepared by completing 5 µL of template DNA, and the final volume to 25 µL sterile deionized water.As reaction cycling conditions using the primers shown in Table 2 were 94 °C for 5 min (1 cycle), 30 cycles of 1 min at 94 °C, 30 s at 60 °C and 1 min at 72 °C, and the final synthesis was used at 72 °C for 5 min.The PCR products and 1 kbp DNA ladder (Thermo, USA) were run on a 1% agarose gel containing 0.5 mg/L ethidium bromide.Then, it was decided whether the expected amplicon sizes were obtained by visualizing them under UV light.
The presence of resistance genes was investigated by polymerase chain reaction (PCR) and at least one or more β-lactamase genes were detected in each sample (Table 5).In addition, more than one resistance gene association was detected in some strains.The rates of resistance genes alone or in combination are as in Tables 6 and 7.The most common bla OXA-48 gene was detected in isolates, and it was detected in fifty-eight (96.6%) of the isolates.
I1 plasmid type was found most frequently among the strains (n = 20, 33.3%).The HI2, K, W, FIC, A/T, T, FIIs, K/B replicon types could not be detected in any of the strains (Table 8).When evaluated in terms of plasmid associations, association was detected in 1 or two strains in 2 and 3 combinations (Table 9).I1 + FIB + Y Table 2 List of primers used in plasmid typing studies [7] Replicon Type Primers (5' → 3')

: T T C T C C T C G T C G C C A A A C T A G A T A/C F: G A G A A C C A A A G A C A A A G A C C T G G A repA X73674 465 R: A C G A C A A A C C T G A A T T G C C T C C T T T F: T T G G C C T G T T T G T G C C T A A A C C A T repA K00053 750 R: C G T T G A T T A C A C T T A G C T T T G G A C FIIs F: C T G T C G T A A G C T G A T G G C repA AE006471 270 R: C T C T G C C A C A A A C T T C A G C F repB F: T G A T C G T T T A A G G A A T T T T G RNAI/repA AY234375 270 R: G A A G A T C A G T C A C A C C A T C C K/B F: G C G G T C C G G A A A G C C A G A
and L/M + Y plasmid associations were mostly found in 2 strains (6.25%).The most common combination of plasmid types was determined as IncI1 + IncFIB + IncY, and IncL/M + IncY.

Conclusion
The emergence of antibiotic-resistant infections is recognized as a serious problem for human health worldwide.The widespread use and misuse of antibiotics has led to a decrease in the effectiveness of conventional antimicrobial therapy and appropriate antibiotic selection [8].In this study, isolation from urine in eighteen (30%) isolates and from blood in eleven (18.3%) isolates show that infections are most related to urinary system and bloodstream infections.According to the 2020 report of the Central Asian and European Surveillance of Antimicrobial Resistance (CAESAR) study, which also includes our country, the rate of carbapenem resistance in K. pneumoniae strains is 43% [10].In this study, carbapenem resistance was found to be 87.2% in 60 K. pneumoniae strains, much higher than the Asian and European averages.All the K. pneumoniae strains examined in our study were found to be ampicillin/sulbactam and ertapenem resistant but colistin susceptible.Amoxacillin/clavulonic acid resistance was detected at 98.14% of strains.This also demonstrates the effects of commonly used antibiotics.The rates of strains carrying bla GES , bla NDM , bla TEM , bla SHV , bla CTX-M1 , bla CTX-M2 , bla IMP , and bla OXA-48 type resistance genes in their isolates were determined.The most common type was bla OXA-48 with 96.6%, followed by bla SHV with 91.6%, bla TEM with 78.3%, bla CTX-M-1 with 63.3%, bla CTX-M-2 with 18.3%, bla IMP with 13.3%, followed 6.6% by bla NDM , and 1.6% by bla GES .
After the bla OXA-48 gene was first detected in K. pneumoniae in 2001, nosocomial outbreaks of bla OXA-48 positive K. pneumoniae have occurred within five years [11].In the study by Ece et al. [12], all K. pneumoniae isolates were carbapenem resistant; all contained bla OXA-48 and all were sensitive to both gentamicin and colistin.In our study, 96.6% of carbapenem-resistant K. pneumoniae isolates contained bla OXA-48 , and all were found to be susceptible to colistin and 61% to gentamicin.The reason for this high rate is that Turkey is the epicenter for bla OXA-48 .
The bla SHV1 gene, which is the precursor of SHV-type β-lactamases, is most common in K. pneumoniae worldwide.Oksuz et al. in Türkiye, 92.9% of the bla SHV and 52.2% of the bla TEM gene was found in K. pneumoniae strains [14].Similarly, in our study, bla SHV was detected with a rate of 91.6% but bla TEM was found in 78.3%.From the point of view of bla CTX-M-1 and bla CTX-M-2 , they found 62-87.7% and 0-100% [15,16].In our study, bla CTX-M-1 was detected in 63.3% and bla CTX-M-2 in 18.3%.These results are within average for the presence of these genes.In terms of the presence of bla NDM and bla IMP genes, it was found to be 6.6% and 13.3% lower in carbapenemresistant K. pneumoniae strains investigated compared to previous studies [13,17].In our study, bla VIM , bla PER , bla VEB , bla KPC , bla OXA-23-24-51-58 gene positive isolates were not detected in any of the 60 isolates.Similarly, bla PER and bla VEB type β-lactamases were not detected in any strain by Bektas et al. in our country [18].In a study conducted by Sağıroğlu et al. [13]. in Türkiye, they found 2% of bla KPC positivity.In our study, however, no bla KPC resistant isolate was detected.This situation gives us concern that the bla KPC resistance gene has not become widespread yet, but that bla KPC resistance will increase in the future.It is thought that the knowledge gained by monitoring the resistance, conducting the necessary studies to prevent the development of resistance, systematically evaluating the data of our country, choosing the antimicrobial treatment correctly and appropriately, preventing unnecessary antibiotic use, ensuring the success of the treatment, and contributing to the country's economy because of these are thought to be valuable.8).Kalaycı-Yuksek et al., in their study conducted in our country in 2023, found that 28 strains containing both bla NDM and bla OXA-48 , IncL/M (7.7%) has determined [19].
In terms of IncY, it was 3.2% in Malaysia and 8.1% in Iran [20,21].The fact that IncY was found to be 30.7% in our study is an indication of the increased spread of IncY plasmids in our country.In a study conducted by Zhou et al. [22]. in China, they found IncI1 to be 1.03%.In the study by Hosny et al. [23] in Egypt, IncI1 was found to be 3.12%.In our study, IncI1 was found to be 76.9%.Our results were found to be quite high compared to other studies.
When we compare with the studies conducted worldwide in terms of IncFIA, IncFIB, IncN, IncP, IncFrepB, and IncL/M types, our present results show that the rate of these types is lower or on the average of the data [20][21][22][23][24]15].In our study, IncHI1 was found to be 3.8%.This is the first record in Türkiye and this may be an indication that IncHI1 has just started to spread in our country.But in our study, IncHI2, IncX, IncW, IncFIC, IncA/C, IncT, Inc-FIIs, IncK/B, and IncB/O plasmid profiles were not found.We believe that this is due to regional differences and the fact that the spread has not yet occurred.
The fact that plasmids were detected in most of the strains suggests that resistance is an indicator of spread by plasmid.In this study, plasmid profiles that we investigated in K. pneumoniae in clinical samples for the first time in our country were examined, and the first data on the frequency of plasmids were presented.In particular, very high rates of IncI1 and IncY plasmid types were detected.The presence of these plasmid profiles in our country brings with it the concern that antibiotic resistance is spreading rapidly and that these plasmid types will be more common in our country in the future.The increase in carbapenem resistance is largely driven by conjugative plasmids.Such plasmids carry multiple or single markers of resistance such as bla NDM and bla OXA-48 [25].
This study provides an overview of the major plasmid families occurring in multiple antibiotic-resistant strains of K. pneumoniae.To our knowledge, this study represents the first report of IncHI1 record in Türkiye.In addition, co-production of NDM-1 and OXA-48   carbapenemases in Türkiye was first performed by Kilic and Baysallar, it is clear that this resistance has increased in Türkiye in the last eight years [26].OXA-48 was detected in all strains except strains 11 and 28.The fact that strains have at least one of the detected plasmid types shows the importance of the presence of plasmids in the rapid spread of OXA-48 and other resistance genes and also explains the increasing resistance rate and difficult treatment processes.

Table 3
Medical units from which K. pneumoniae isolates are obtained

Table 6
Distribution and gene associations of extended spectrum beta lactamase (ESBL) genes detected in K. pneumoniae isolates (n = 60)

Table 7
Gene associations of ESBL genes in strains (n = 60)Note: Only those with resistance gene are marked as "+".Samples without resistance gene were not labeled

Table 8
Plasmid detected strains and their detected plasmid types Note:The strain for which no replicon type was identified is not shown in the table.

Table 9
Plasmids co-occurrence combinations and their rates * Percentages in the number of cases were taken according to the number of strains (n = 32) with the plasmid detected